Recent accomplishments include the following: [] ANALYSES OF FUNCTIONS OF AN ANTI-PD-L1/TGFbetaR2 BISPECIFIC FUSION PROTEIN (M7824). M7824 (MSB0011359C) is a novel first-in-class bifunctional fusion protein consisting of a fully human IgG1 anti-PD-L1 monoclonal antibody (with structural similarities to avelumab) linked to the extracellular domain of two TGFbeta receptor 2 (TGFbetaR2) molecules serving as a TGFbeta Trap. Avelumab has demonstrated clinical activity in a range of human cancers and has been approved by the FDA for the therapy of Merkel cell and bladder carcinomas. Preclinical studies have shown this anti-PD-L1 is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). In the studies reported here, it is shown that M7824 is also capable of mediating ADCC of a wide range of human carcinoma cells in vitro, employing natural killer (NK) cells as effectors, albeit not as potent as anti-PD-L1 employing some tumor cells as targets. The addition of the IL-15 superagonist fusion protein complex ALT-803 enhanced the ADCC capacity of both anti-PD-L1 and M7824, and to levels that both agents now demonstrated similar levels of ADCC of tumor cells. TGFbeta is a known immunosuppressive entity. Studies reported here show TGFbeta1 induced reduction of several NK activation markers as well as reduction of endogenous NK lytic activity and NK-mediated ADCC of tumor cells. These phenomena could be reduced or mitigated, however, by M7824, but not by anti-PD-L1. M7824, but not anti-PD-L1, was also shown to reduce the immunosuppressive activity of regulatory T cells on human CD4+ T-cell proliferation. These studies thus demonstrate the dual functionalities of M7824 and provide the rationale for its further clinical development. [] M7824, A NOVEL BIFUNCTIONAL ANTI-PD-L1/TGFBETA TRAP FUSION PROTEIN, PROMOTES ANTI-TUMOR EFFICACY AS MONOTHERAPY AND IN COMBINATION WITH VACCINE. Tumors evade host immune surveillance through multiple mechanisms, including the generation of a tumor microenvironment that suppresses immune effector function. Secretion of TGFbeta and upregulation of immune checkpoint programmed cell death ligand-1 (PD-L1) are two main contributors to immune evasion and tumor progression. We examined the efficacy of a first-in-class bifunctional checkpoint inhibitor, the fusion protein M7824, comprising the extracellular domain of human TGFbetaRII (TGFbeta Trap) linked to the C-terminus of human anti-PD-L1 heavy chain (aPD-L1). We demonstrate that M7824 reduces plasma TGFb1, binds to PD-L1 in the tumor, and decreases TGFbeta-induced signaling in the tumor microenvironment in mice. In murine breast and colon carcinoma models, M7824 decreased tumor burden and increased overall survival as compared to targeting TGFbeta alone. M7824 treatment promoted CD8+ T cell and NK cell activation, and both of these immune populations were required for optimal M7824-mediated tumor control. M7824 was superior to TGFbeta- or aPD-L1-targeted therapies when in combination with a therapeutic cancer vaccine. These findings demonstrate the value of using M7824 to simultaneously target TGFbeta and PD-L1/PD-1 immunosuppressive pathways to promote anti-tumor responses and efficacy. The studies also support the potential clinical use of M7824 as a monotherapy or in combination with other immunotherapies, such as therapeutic cancer vaccines, including for patients who have progressed on aPD-L1/aPD-1 checkpoint blockade therapies. [] ADCC EMPLOYING AN NK CELL LINE (haNK) EXPRESSING THE HIGH AFFINITY CD16 ALLELE WITH AVELUMAB, AN ANTI-PD-L1 ANTIBODY. NK-92 cells, and their derivative, designated aNK, were obtained from a patient with non-Hodgkin lymphoma. Prior clinical studies employing adoptively transferred irradiated aNK cells have provided evidence of clinical benefit and an acceptable safety profile. aNK cells have now been engineered to express IL-2 and the high affinity (ha) CD16 allele (designated haNK). Avelumab is a humanIgG1 anti-PD-L1 monoclonal antibody, which has shown evidence of clinical activity in a range of human tumors. Prior in vitro studies have shown that avelumab has the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of human tumor cells when combined with NK cells. In studies, the ability of avelumab to enhance the lysis of a range of human carcinoma cells by irradiated haNK cells via the ADCC mechanism is demonstrated; this ADCC is shown to be inhibited by anti-CD16 blocking antibody and by concanamycin A, indicating the use of the granzyme/perforin pathway in tumor cell lysis. Avelumab-mediated lysis of tumor cells by irradiated haNK cells is also shown to be similar to that of NK cells bearing the V/V Fc receptor high affinity allele. These studies thus provide the rationale for the clinical evaluation of the combined use of avelumab with that of irradiated adoptively transferred haNK cells. [] IDENTIFICATION AND CHARACTERIZATION OF ENHANCER AGONIST HUMAN CYTOTOXIC T-CELL EPITOPES OF THE HUMAN PAPILLOMAVIRUS TYPE 16 (HPV16) E6/E7. Human papillomavirus (HPV) is associated with the etiology of cervical carcinoma, head and neck squamous cell carcinoma, and several other cancer types. Vaccines directed against HPV virus-like particles and coat proteins have been extremely successful in the prevention of cervical cancer through the activation of host HPV-specific antibody responses; however, HPV-associated cancers remain a major public health problem. The development of a therapeutic vaccine will require the generation of T-cell responses directed against early HPV proteins (E6/E7) expressed in HPV-infected tumor cells. No HPV therapeutic vaccine has been approved by the Food and Drug Administration to date. One method of enhancing the potential efficacy of a therapeutic vaccine is the generation of agonist epitopes. We reported the first description of enhancer cytotoxic T lymphocyte agonist epitopes for HPV E6 and E7. Agonist epitopes have potential for use in a range of HPV therapeutic vaccine platforms. [] ENHANCED ANTITUMOR EFFECTS BY COMBINING AN IL-12/ANTI-DNA FUSION PROTEIN WITH AVELUMAB, AN ANTI-PD-L1 ANTIBODY. The combined therapeutic potential of an immunocytokine designed to deliver IL-12 to the necrotic regions of solid tumors with an anti-PD-L1 antibody that disrupts the immunosuppressive PD-1/PD-L1 axis yielded a combinatorial benefit in multiple murine tumor models. The murine version of the immunocytokine, NHS-muIL12, consists of an antibody (NHS76) recognizing DNA/DNA-histone complexes, fused with two molecules of murine IL-12 (NHS-muIL12). By its recognition of exposed DNA, NHS-muIL12 targets IL-12 to the necrotic portions of tumors; it has a longer plasma half-life and better antitumor efficacy against murine tumors than recombinant murine IL-12. NHS-muIL12, in an IFN-gamma dependent mechanism, upregulates mPD-L1 expression on mouse tumors. Concurrent therapy with NHS-muIL12 and an anti- PD-L1 antibody resulted in additive/synergistic antitumor effects in PD-L1-expressing subcutaneously transplanted tumors and in an intravesical bladder tumor model. Antitumor efficacy correlated with (a) with a higher frequency of tumor antigen-specific splenic CD8+ T cells and (b) enhanced T cell activation over a wide range of NHS-muIL12 concentrations. These findings suggest that combining NHS-muIL12 and an anti-PD-L1 antibody enhances T cell activation and T cell effector functions within the tumor microenvironment, significantly improving overall tumor regression. These results should provide the rationale to examine the combination of these agents in clinical studies.